Project description:Some phages and microbes employ Diversity-Generating Retroelements (DGRs) to achieve rapid targeted adaptation. DGRs mutagenize a specific gene without harming the rest of the genome. Mutagenesis relies on the DGR reverse transcriptase (dRT), which is highly error-prone when transcribing adenines of an RNA template. The resulting mutagenized cDNA then alters the target gene through a poorly understood mechanism. Transplanting DGRs into a tractable organism, like Escherichia coli, holds promise for creating a powerful system for gene-targeted editing. Here, we successfully reconstituted the archetypal BPP DGR in E. coli, demonstrating that DGRs are "plug-and-play" systems adaptable to non-native hosts. Systematic analysis of reconstituted DGR established principles for target mutagenesis. Our results revealed that cytosine misincorporation is the most common error when dRT transcribes adenines. However, error rates peak in 5'-AAC-3' and 5'-ACC-3' contexts, driven by elevated adenine and guanosine misincorporations, respectively. Using high-throughput genetics, we discovered that cells lacking the single-stranded DNA exonuclease ExoI exhibit 15-fold higher DGR activity. Our evidence indicates that ExoI inhibits DGRs by binding a limited cellular factor, rather than through direct cDNA degradation. Finally, by profiling DGR targets across thousands of chromosomal loci, we discovered that DGRs preferentially edit targets near the replication origin and oriented in the same direction as replication. We showed that the directionality of replication underlies this bias, possibly through unwinding the target to facilitate base-pairing with incoming cDNA. These findings establish a reconstituted DGR system in E. coli and provide a foundation for optimizing targeted gene-editing applications in the future.
Project description:Azole resistance was induced in vitro by growth of a susceptible C. parapsilosis isolate in the presence of voriconazole. Whole genome microarrays were used to compare the transcriptional response of the voriconizole-resistant and susceptible isolates.
Project description:Azole resistance was induced in vitro by growth of a susceptible C. parapsilosis isolate in the presence of posaconazole. Whole genome microarrays were used to compare the transcriptional response of the posaconazole-resistant and susceptible isolates.
Project description:Azole resistance was induced in vitro by growth of a susceptible C. parapsilosis isolate in the presence of fluconazole. Whole genome microarrays were used to compare the transcriptional response of the fluconazole-resistant and susceptible isolates.
Project description:The present study describes a novel mechanism of antifungal resistance affecting the susceptibility of both the azole and echinocandin antifungals in an azole-resistant isolate from a matched pair of C. parapsilosis isolates obtained from a patient with prosthetic valve endocarditis. Transcriptome analysis indicated differential expression of several genes in the resistant isolate including upregulation of ERG1, ERG2, ERG5, ERG6, ERG11, ERG24, ERG25, ERG27, DAP1 and UPC2, of the ergosterol biosynthesis pathway. Whole genome sequencing revealed a mutation in the ERG3 gene leading to a G111R amino acid substitution in the resistant isolate. Subsequent introduction of this allele in the native ERG3 locus in the susceptible isolate resulted in a fluconazole MIC of >64 mg/ml and a caspofungin MIC of 8 mg/ml. Corresponding allelic replacement of the wildtype allele for the mutant allele in the resistant isolate resulted in a drop in MIC to 1 mg/ml for both fluconazole and caspofungin. Sterol profiles indicated a loss of sterol demethylase activity as a result of this mutation. This work demonstrate that this G111R mutation is wholly responsible for the resistant phenotype in the C. parapsilosis resistant isolate and is the first report of this multidrug resistance mechanism.
Project description:description Blastocystis sp. is a highly prevalent anaerobic eukaryotic parasite of humans and animals. The genome of several representatives has been sequenced revealing specific traits such as an intriguing 3’-end processing of primary transcripts. We have acquired a first high-throughput proteomics dataset on the difficult to cultivate ST4 isolate WR1 and detected 2,761 proteins. We evidenced for the first time by proteogenomics a functional termination codon derived from transcript polyadenylation for seven different key cellular components.
